The energy-transducing ATPase complex in membranes of Escherichia coli is a reversible, proton-transducing ATPase. It catalyzes the transphosphorylation reaction of oxidative-phosphorylation, utilizing an electrochemical gradient of protons delta micron H ion generated by electron transport. The complex can be disassociated into two entities: the F-ATPase which retains ATP hydrolytic activity, and the F0-sector which catalyzes the H ion 1 translocation across the membrane. The objective of the proposed project is to identify the polypeptide subunits of the F0-sector and determine the specific role of each subunit in F0 function. The problem will be approached by both biochemical and genetic analysis. The F1F0-complex will be purified in order to identify the putative subunits of F0. The F0-sector will then be disassociated from F1 and the proton-translocating activity will be reconstituted in phospholipid liposomes. In order to determine which subunits of F0 function in proton-translocation, a series of mutants with inactivated H plus -translocases will be isolated and analyzed. By genetic complementation we will determine the number of alleles affecting proton-translocase activity. We hope to identify the altered subunits in these mutants by contrasting them to wild-type subunits on dodecyl sulfate electrophoresis and/or isoelectric focusing. In this way subunits of the purified complex with a role in proton-translocation will be distinguished from those that may be adventiously associated during purification. Ultimately we would like to purify each of the functionally implicated subunits and reconstitute proton-translocation from the minimal number of subunits. By studying the structure of these subunits, and the structure of F0 in the membrane, we hope to gain insight into the mechanism of F0 -mediated proton-translocation.